Recent years have witnessed a welcome explosion in the study and development of topical microbicides to prevent HIV transmission. Among the important recent developments are included contributions of members of this team. Developing an amino-terminus modified RANTES analogue, PSC-RANTES, which is several logs more potent than the native RANTES and is so far the only agent shown to afford high level protection (10/10 animals) from SHIV in the rhesus vaginal challenge model, we have demonstrated that CCR5 blockade alone is sufficient to block SHIV transmission in this system. Nonetheless, current costs of synthesis may render this reagent is too expensive for use at the highest, most effective concentrations. At the same time, one member of our team has demonstrated that combinations of microbicide candidates can synergize to provide high level protection in the rhesus challenge model. We therefore propose in this application a series of high risk, exploratory studies to test the rationale for combination microbicide strategies by testing the following hypotheses: 1.Cellular activation at mucosal sites of HIV entry promotes HIV dissemination and a topical immune modulator can antagonize activation and provide synergistic activity with lower doses of PSC-RANTES. This is plausible since there is increasing evidence that cellular activation is a critical underpinning of HIV pathogenesis, of viral dissemination from mucosal sites during early infection and possibly also in susceptibility to HIV infection. 2. Topical application of type 1 interferons will block HIV replication without the topical inflammation induced by TLR stimulation and this activity will synergize with CCR5 blockade as induced by PSC-RANTES. This is a plausible strategy as interferons comprise a major arm of innate host defenses against viral infection, are suppressive of HIV replication and also play a role in the arming of other innate and adaptive defense mechanisms. 3. Topical application of immune modulators will provide synergistic antiviral activity when co-administered with a type I interferon as the latter will block HIV infection of target cells and arm both innate and adaptive immune defenses while the former will block dissemination of infection that is dependent upon immune activation. These hypotheses will be tested in PBMC, in epidermal Langerhans cells and in ectocervical explants. If warranted by results in these systems, a promising combination strategy will be selected for further testing in the rhesus vaginal challenge model with concurrent studies of protective activity, safety and effects on immunologic indices and microbiologic indices of topical immune suppression. We expect that the results of these studies will provide insights into mechanisms of HIV acquisition across mucosal surfaces that will guide the further development of topical strategies to prevent HIV transmission. [unreadable] [unreadable] [unreadable]